Decreased insulin action, impaired insulin secretion and increased adiposity are important risk factors for development of type 2 diabetes. These risk factors are present even when individuals have both normal fasting and two hour glucose concentrations indicating a very early role for decreased insulin secretion in the development of type 2 diabetes. In previous work we found that a a bimodally expressed gene in muscle tissue. HLA-DRB1, was associated with lower risk for diabetes and higher insulin secretion in Pima Indians. As this protective HLA haplotype system indicates involvement of autoimmunity in progression to type 2 diabetes in Pima Indians we performed a pilot study in individuals with and without the HLA-DRB1 locus who were also discordant for diabetes status, examining both immunoprofiling of over 9,000 potential proteins and T-cell receptor genes. From this study we have selected 80 antibodies with significantly increased concentrations in those with diabetes to investigate in a larger co-hort discordant for diabetes and the protective haplotype. . We also sequenced the complementary defining region 3 (CDR3) in the T cell receptor in these same individuals. We found that the length of the CDR3 region is shorter in those with diabetes indicating loss of self tolerance. We have subsequently sequenced CDR3 regions in a larger number of individuals without the protective HLA haplotype who also have measures of insulin action and secretion. We are continuing to investigate additional factors which control insulin secretion and insulin action. Individuals without diabetes who are undergoing bariatric surgery using the following techniques: Roux-en-Y gastric bypass (RYGBP), laporoscopic band, or gastric sleeve have undergone measures of insulin action, insulin secretion and meal tests prior to and one month following the surgery. Results indicate that individuals who underwent RYGBP had improvements in insulin action hepatic insulin sensitivity, and more rapid glucose and insulin responses during meal tests. This explains the greater improvement in glycemia seen in those with diabetes undergoing RYGBP compared with other the laporoscopic band. Although glucagon like peptide-1 (GLP-1) also increased more during the meal in those who underwent RYGBP, the timing did not indicate that this increase played a role in the increase insulin release. Other gastrointestinal peptides did not differ between the groups except for pancreatic polypeptide, a marker of vagal nerve activity, which decreased significantly during the meal following RYGBP. We are continuing to follow these individuals to examine body composition changes with relation, and determine if the glucose and insulin responses during the meals tests change with further weight loss. Previous predictors of weight gain based on this study have included, higher respiratory quotient, higher insulin mediated glucose uptake, lower free T3, and relatively lower energy expenditure. Variability in energy expenditure is a mediator of weight change, and we have continued to evaluate factors related to metabolic rate. We confirmed this association between lower energy expenditure relative to body size as a predictor of weight gain in a larger cohort with longer follow-up. Furthermore, we were able to demonstrate that lower energy expenditure also predicted gain in fat mass. The thermic effect of food and the energy cost of arousal are overlapping and difficult to measure components of energy expenditure. By examining timepoint data from our metabolic chambers, we were able to estimate these components (which we termed awake fed thermogenesis (AFT)) which accounted for approximately 10% of total energy expenditure. We found that lower AFT predicted weight gain, but only in individuals with BMI 29 kg/m. This indicates that increased insulation associated with adiposity leads to lower cost of metabolizing macronutrients. We found that very long chain fatty acid concentrations in the CSF were inversely associated with 24 hour energy expenditure indicating a role for specific long chain fatty acids as central signaling molecules with important peripheral effects. We are planning a follow-up study in which we administer a specific long chain fatty acid and determines its effect on metabolic parameters. We have also confirmed the role of relatively lower 24 hour energy expenditure as a predictor of weight and fat mass gain. Genetic factors underlie adiposity and its risk factors. Mutations in the melanocortin 4 receptor gene are associated with increased body mass index and lower 24 hour energy expenditure in humans. Inidividuals with MC4R mutations have accelerated weight gain in childhood but not in adulthood indicating a more potent effect of this mutation in early life. Furthermore, we found that presence of MC4R mutations predicted development of diabetes in childhood independent of body weight. The mechanism by which MC4R leads to hyperphagia and weight gain is not clear. One possible mediator is brain derived neurotrophic factor (BDNF) a downstream effector of MC4R signaling which has been implicated in childhood hyperphagia and weight gain. However, serum BDNF did not differ between individuals with and without MC4R mutations.